Stem cells isolated from human exfoliated deciduous teeth (SHEDs) are a type of mesenchymal stem cells (MSCs), widely investigated for regenerative treatment. They are isolated from dental pulp tissues remaining in physiologically shedding human deciduous teeth. Thus, SHEDs are easy to access and not required invasive procedure to obtain cells. SHEDs are multipotent mesenchymal stem cells; however, they possess distinct properties when compared to other MSCs. In this regard, SHEDs exhibit higher proliferative rate than bone marrow‐derived MSCs and greater osteogenic differentiation potency than human dental pulp stem cells. This chapter reviews the isolation technique and basic characteristics of SHEDs. Moreover, the intracellular signalling involved in the stemness regulation and differentiation ability of SHEDs is discussed, particularly on fibroblast growth factor, Notch, and Wnt signalling. Finally, the potential regenerative therapeutic application of SHEDs is also described.
- stem cells
- deciduous teeth
- basic fibroblast growth factor
- Wnt signalling
- Notch signalling
- mechanical stress
Dental pulp is a loose connective tissue residing in pulp chamber inside both deciduous and permanent teeth. It surrounds by hard tissues called dentin. Nutrients and oxygen supply are acquired from blood vessels passing through apical and accessory foramen of the teeth’s root. Dental pulp originates from cranial neural crest cells . Dental pulp tissues are composed of extracellular matrix and various cell types, e.g. fibroblasts, odontoblasts, endothelial cells, pericytes, immune cells and stem cells. When injured, cells in dental pulp tissues are capable of differentiating odontoblasts or odontoblast‐like cells, leading to the promotion of tertiary dentin formation. The formation of tertiary dentin is a mechanism which can protect the tooth vitality. Dental pulp tissues remaining in physiological shedding of deciduous teeth are the alternative source of mesenchymal stem cells, due to the ease of accessibility and minimally invasive technique to obtain tissues . Stem cells from human exfoliated deciduous teeth (SHEDs) are firstly identified by Miura et al. in 2003 . SHEDs have high proliferation potency and are multipotent mesenchymal stem cells. These cells are able to differentiate into, not only, dental pulp‐related cells, but also, other cell lineages, for example osteoblasts, adipocytes, neuronal‐like cells and endothelial cells [2–8]. Taking these advantageous properties together, SHEDs are one of the candidate cell types for tissue regeneration study.
2. SHEDs’ characteristics
SHEDs are heterogeneous population of cells isolated from dental pulp tissues remained in exfoliated deciduous teeth. Similar to those mesenchymal stem cells (MSCs), SHEDs exhibit fibroblast‐like morphology, adhere on plastic tissue culture surface, express mesenchymal stem cell surface marker and have multipotential differentiation ability (Figure 1). SHEDs have higher proliferation rate compared to dental pulp stem cells (DPSCs) and bone marrow‐derived mesenchymal stem cells (BMMSCs) [2, 9]. This could be due to the high expression of genes related to cell proliferation and extracellular matrix in SHEDs comparing with DPSCs . First, a study by Miura et al. demonstrated that SHEDs express mesenchymal surface markers, STRO‐1 and CD146 , though, the percentage of positive cells is low . Later studies utilized various surface markers for SHEDs characterization protocol. SHEDs expressed CD44, CD73, CD90, CD105 and STRO‐1 . In addition, these cells lack of CD45 expression . Besides these markers described above, SHEDs also express other surface markers for example, CD166 and SSEA4. Lack of CD34 is also reported . There is no specific surface marker to precisely identify SHEDs population.
Up to date, MSCs can be isolated from many tissue types. Though, there is no specific marker to clearly identify these cells. According to the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy, the minimum criteria to identify MSCs are as follow . First, the isolated MSCs should adhere to plastic tissue culture plate . Second, MSCs must express several specific surface markers, namely CD105, CD73 and CD90 . They also should not express CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA‐DR . Finally, MSCs have to be able to differentiate into osteoblasts, adipocytes and chondroblasts
2.1. Isolation technique
Two methods have been utilized for SHEDs isolation, namely an enzymatic digestion and a tissue explant. The enzymatic digestion is performed by digesting minced remaining pulp tissues from deciduous teeth, normally with type I collagenase and dispase mixed enzyme solution [12–14]. For tissue explant, minced pulp tissues are placed on the tissue culture dishes, allowing the outgrowth of the cells from the tissues . Enzymatic digestion technique leads to more heterogeneous population of isolated cells than those obtained from tissue outgrowth protocol . A study illustrated that there is no significant difference regarding cell morphology and proliferation between cells isolated using enzymatic digestion and tissue outgrowth . Enzymatic digestion‐derived SHEDs had higher mineralization ability
2.2. Differentiation potential of SHEDs
Studies have shown that SHEDs possess multi‐differentiation potency similar to MSCs. Those lineages include odontogenic/osteoblastic, adipogenic, neurogenic and angiogenic differentiation .
2.2.1. Odontogenic/osteoblastic differentiation potential
The ability of SHEDs to differentiate into odontoblastic lineage is widely known [2, 15, 16]. Primitively, SHEDs were characterized by their
Evidence suggested that SHEDs might have the preference towards the odontoblastic lineage due to its origin. SHEDs can be induced to become functional odontoblasts
2.2.2. Neurogenic differentiation potential
Neurogenic potential of SHEDs is expecting due to their neural crest embryonic origin. Several research studies focusing on differentiating dental stem cells to be used for neurodegenerative disease therapy. These cells are prone to undergo neurogenic differentiation both
2.2.3. Angiogenic differentiation potential
Angiogenic potential of SHEDs is another aspect of interest for the benefit of connective tissue regeneration. The rapid and effective induction of vasculation is required for sufficiently supply of oxygen and nutrients as well as removing the toxic waste from the newly synthesized tissues. Unstimulated SHEDs expressed VEGFR1 and NP‐1, the known important receptors in angiogenesis and VEGFR1 signalling play an important role in VEGF‐induced capillary tube formation by SHEDs as shown by VEGFR1 gene silencing . SHEDs cultured in the tooth slice/scaffolds in combine with VEGF expressed several endothelial differentiation markers such as VEGFR1, VEGFR2, platelet endothelial cell adhesion molecule‐1 (PECAM‐1) and vascular endothelial cadherin (VE‐Cadherin). When transplanted in immunodeficient mice, SHEDs actually lined the new blood vessels within the tooth slice/scaffolds close to the blood vessels of host . Similar results were observed when SHEDs seeded in human tooth slice/scaffolds and transplanted into immunodeficient mice differentiate into human blood vessels that anastomosed with the mouse vasculature and VEGF induced the angiogenic differentiation of SHEDs through Wnt/β‐catenin signalling . Another study also showed that SHEDs can differentiate into VEGFR2‐positive and CD31‐positive endothelial cells
2.2.4. Adipogenic differentiation potential
Several studies have reported that SHEDs can be induced into adipogenic lineage [6, 32–34]. After cultured in an adipogenic medium, SHEDs’ morphology changed from spindle‐like to polygonal shapes and lipid vacuoles were observed, along with the increased in PPARγ2 and LPL mRNA . However, the studies evaluated the adipogenic potential of SHEDs
2.3. Immunomodulatory property
Like other MSCs, SHEDs exhibit immunomodulatory properties. Though, the potency and mechanism are not exact the same to those of BMMSCs [10, 35]. SHEDs significantly reduced the percentage of IL17+IFNγ cells population in CD4+ T cells
3. Basic fibroblast growth factor signalling in SHEDs
Basic fibroblast growth factor (bFGF) is a member in fibroblast growth factor family . It binds to fibroblast growth factor receptors (FGFR) and further initiates intracellular signalling . bFGF has been shown to participate in the regulation of stemness maintenance and cellular differentiation. In human DPSCs, bFGF promotes pluripotent stem cell marker expression, corresponding with the increase of colony‐forming unit . Furthermore, bFGF inhibits osteogenic differentiation by SHEDs, human DPSCs and human periodontal ligament stem cells (PDLSCs) when supplemented in osteogenic induction medium (Figure 2) [5, 40]. In this regard, alkaline phosphatase enzymatic activity and mineralization are markedly decreased under bFGF‐treated condition compared with the control [5, 40]. On the contrary, bFGF enhances the expression of neurogenic marker, βIII‐tubulin, via FGFR and PLCγ when human DPSCs are cultured in a neurogenic induction medium supplemented with bFGF .
In SHEDs, long‐term culture
Regarding osteogenic differentiation, bFGF attenuated osteogenic differentiation. In this regard, bFGF attenuated alkaline phosphatase enzymatic activity and mineralization in SHEDs after osteogenic induction [5, 43]. The inhibition of endogenous bFGF in SHEDs either by a chemical inhibitor for FGFR or lentiviral shRNA against bFGF resulted in the enhancement of osteogenic differentiation . It was also demonstrated that bFGF attenuated alkaline phosphatase mRNA expression and mineral deposition via FGFR and MEK signalling pathway .
Several possible mechanisms were reported. Firstly, bFGF might attenuate osteogenic differentiation in SHEDs via decreasing Notch signalling . Notch signalling activation led to the enhancement of mineralization in SHEDs . Treatment with bFGF attenuated Notch receptor, ligand and target gene expression which may participate in bFGF attenuated osteogenic differentiation in SHEDs . Secondly, bFGF inhibited matrix metalloproteinase (MMP) expression, for example
4. Wnt signalling in SHEDs
Canonical Wnt signalling also has a significant role in tooth development and stem cells self‐renewal through β‐catenin [46, 47]. Inactivation of β‐catenin in the mesenchyme of developing tooth results in arrested tooth developmental at the bud stage . Various studies established the influence of canonical Wnt signalling pathway to promote the osteogenic differentiation of dental stem cells, i.e. DPSCs, PDLSCs, stem cells from apical papilla (SCAPs) and dental follicle stem cells (DFSCs) [49–52]. However, the effect of the canonical Wnt/ β‐catenin on SHEDs is very limited. The involvement of Wnt/β‐catenin on SHEDs‐mediated mineralized tissue regeneration was investigated with the addition of basic fibroblast growth factor (bFGF) . Treatment with bFGF attenuated SHEDs‐mediated mineralized tissue regeneration via activation of ERK 1/2 pathway and consequently inhibited Wnt/β‐catenin pathway, leading to osteogenic deficiency of SHEDs .
Activation of β‐catenin by LiCl in SHEDs led to the significant decrease of colony formation by SHEDs . In addition, LiCl enhanced subG0 population in SHEDs .
5. Notch signalling in SHEDs
Notch signalling controls various function of stem cells, ranging from stemness maintenance to cell‐specific differentiation . It is a highly conserved pathway, firstly identified in Drosophila. Notch signalling is initiated by the binding between membrane‐bound Notch receptors and ligands of neighbouring cells [56–58]. Further, Notch receptors are cleaved by a γ‐secretase enzyme, leading to the release of Notch intracellular domain (NICD) [56–58]. Subsequently, NICD translocates into nucleus and forms complex with other transcriptional molecules, resulting in the activation of Notch target genes [56–58]. Common Notch signalling target genes are Hes and Hey families [56–58]. In the canonical Notch signalling pathway, four receptors and five ligands are identified [56–58]. The four types of Notch receptors are Notch1, Notch2, Notch3 and Notch4. Five ligands are Delta‐like‐1 (Dll‐1), Delta‐like‐3 (Dll‐3), Delta‐like‐4 (Dll‐4), Jagged1 and Jagged2 [56–58].
Notch signalling participates in odontogenesis, dental pulp repair and regeneration. Mice lacking of Jagged2 expression exhibited defective enamel formation of incisors and malformation of molars . The expression of Notch receptors and ligands was upregulated in response to calcium hydroxide, a material for direct pulp capping treatment . Human DPSCs over‐expressing Jagged1 exhibited the reduction of osteogenic differentiation ability and mineralization
Studies illustrated that indirectly immobilized Notch ligands, Jagged1 or Dll‐1, on tissue culture surface increased
It has been shown that bFGF inhibited the mRNA expression of Notch signalling components. In this regard, bFGF significantly reduced the mRNA levels of
6. Mechanical stress influences SHEDs’ behaviours
Dental pulp tissues are surrounded by hard tissues, namely dentin. During inflammation, an interstitial fluid pressure increases [65, 66], causing biological changes in local cells and tissues. In addition, fluid movement in dentin‐pulp complex during normal occlusal force may expose cells to mechanical stimuli . Mechanical forces are shown to regulate biological functions in many cell types, for example osteoblasts, osteocytes, periodontal ligament cells and dental pulp cells. Different types and magnitude of force lead to different cell responses. In human DPSCs, uniaxial cycle stretching inhibited odonto/osteogenic differentiation but increased cell proliferation [68, 69], while cyclic hydrostatic pressure synergistically enhanced BMP‐2‐induced DSPP expression by human DPSCs
7. Potential application of SHEDs in regenerative therapy
SHEDs are the good candidate for the stem cells used in regenerative therapy due to their high plasticity as well as ability to cross lineage boundaries and differentiate into several specialized cells. Current progresses have been made for tissue engineering‐based therapies involving a large number of tissues. However, dentin-pulp complex and neuronal tissue seem to be the most promising aspects for the application of SHEDs in regenerative therapy.
The first evidence to show that SHEDs can differentiate to become the functional odontoblasts with the ability to generate the mineralized tissue resemble to dentin was shown in mice . SHEDs were seeded within a scaffold in a tooth slice and implanted into the dorsum of mice. Dental pulp‐like tissue was observed in the central area of the pulp chamber of the tooth slice . The expression of odontoblastic differentiation markers such as DSPP and DMP‐1 was detected . Remarkably, the newly deposited dentin was observed and suggested that SHEDs can differentiate into fully functional odontoblasts
In addition to dentin-pulp complex regeneration, SHEDs also show the potential to be used in neuroregeneration. Stem cell therapy is the promising therapeutic options for treating the neurodegenerative diseases due to the limited regenerative capacity of the specialized cells in the nervous system. The neural crest cell in origin makes SHEDs the candidate cell model for neuron tissue regeneration. These cells are prone to undergo neurogenic differentiation both
In a focal cerebral ischemia rat model induced by permanent middle cerebral artery occlusion, intranasal administration of supernatants from the medium used to culture SHEDs significant decreased in the motor disability score and significantly reduced in the infarct volume . Moreover, positive signals for neuronal nucleus, neurofilament H, doublecortin and rat endothelial cell antigen in the peri‐infarct area were observed in the rats treated with SHEDs conditioned media compared to the DMEM control from approximately 140 mm3 in DMEM control to 50 mm3 in SHEDs conditioned medium . These results suggest that SHEDs might secrete some compounds that positively influence the recovery of the brain lesion in focal cerebral ischemia .
Studies have shown that SHEDs have remarkable neuroregenerative activity and promote functional recovery in a spinal cord injury animal model [29, 75]. Rats that received SHEDs transplantation within the lesion created at the 9th–11th thoracic vertebral levels exhibited higher scores in the locomotor rating scale compared to the bone marrow stromal cells or fibroblasts transplantation control . In addition, the rescue of hindlimb locomotor function was prominent in the rats that received SHEDs. These animals were able to move hindlimb coordinately and walk, while the bone marrow stromal cells transplantation exhibited only subtle movements . A similar trend was observed in another study, a complete recovery of hindlimb motor function was observed after implantation of neural‐induced SHEDs in a rat spinal cord injury  which suggested that preinduction of the undifferentiated SHEDs into the neural‐like cells before implantation might improve the efficiency of SHEDs in regenerating specialized neural cells. Taken together, these high neurogenic potential of SHEDs especially in animal models makes them the favourable source for stem cell regeneration treatment for neural diseases.
Dental stem cells, including SHEDs, have been extensively studied in the past decades leading to the better understanding in their unique biological properties and therapeutic potential. As SHEDs can be easily obtained with limited ethical concern, their multi‐differentiation potentials have been demonstrated, which creates great opportunities for the application in the regenerative therapy. However, despite the intriguing results, we still need further study to deepen the understanding of the mechanisms underlying the differentiation processes to attain clinical reality. Also, the potential risks for the clinically use of SHEDs or other dental stem cells should be thoroughly studied for the safety of the patients who will greatly benefit from their regenerative ability.
The authors thank for support of the Faculty of Dentistry Research Fund, Chulalongkorn University. We would like to thank Dr. Pattarin Potisomporn for the illustration in Figure 4.
|Akt||Protein kinase B|
|bFGF||Basic fibroblast growth factor|
|BMMSCs||Bone marrow‐derived mesenchymal stem cells|
|CD||Cluster of differentiation|
|COL1||Collagen type 1|
|DAPT||N‐[N‐(3,5‐Difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester|
|DFSCs||Dental follicle stem cells|
|DMEM||Dulbecco’s Modified Eagle Medium|
|DMP‐1||Dentin matrix acidic phosphoprotein 1|
|DMP||Dentin matrix protein|
|DPSCs||Dental pulp stem cells|
|ERK||Extracellular signal‐regulated kinase|
|FGFR||Fibroblast growth factor receptor|
|GFAP||Glial fibrillary acidic protein|
|Hes||Hairy and enhancer of split|
|Hey||Hairy and enhancer of split related with YRPW motif protein|
|HGF||Hepatocyte growth factor|
|HLA‐DR||Human leukocyte antigen‐antigen D related|
|LEF‐1||Lymphoid enhancer binding factor 1|
|MEK||Mitogen‐activated protein kinase kinase|
|MEPE||Matrix extracellular phosphoglycoprotein|
|MSCs||Mesenchymal stem cells|
|MSX2||Msh homeobox 2|
|MT1‐MMP||Membrane type1‐ matrix metalloproteinase|
|NICD||Notch intracellular domain|
|Nurr1||Nuclear receptor related 1 protein|
|OCT4||Octamer‐binding transcription factor 4|
|P2Y1||Purinergic receptor P2Y1|
|PCR||Polymerase chain reaction|
|PDLSCs||Periodontal ligament stem cells|
|PECAM‐1||Platelet endothelial cell adhesion molecule 1|
|Pitx3||Paired like homeodomain 3|
|PLCγ||Phospholipase C gamma|
|PPARγ2||Peroxisome proliferator‐activated receptor‐gamma 2|
|REX1||Reduced Expression Protein 1|
|RUNX2||Runt‐related transcription factor 2|
|SCAPs||Stem cells from apical papilla|
|SHEDs||Stem cells isolated from human exfoliated deciduous teeth|
|shRNA||Short hairpin ribonucleic acid|
|SOX2||Sex determining region Y‐box 2|
|SSEA4||Stage‐specific embryonic antigen‐4|
|TERT||Telomerase reverse transcriptase|
|TWIST||Twist Family BHLH Transcription Factor|
|VE‐Cadherin||Vascular endothelial cadherin|
|VEGF||Vascular endothelial growth factor|
|VEGFR||Vascular endothelial growth factor receptor|
Chai Y, Jiang X, Ito Y, Bringas P Jr., Han J, Rowitch DH, Soriano P, McMahon AP, Sucov HM. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development. 2000; 127:1671-9.
Miura M, Gronthos S, Zhao M, Lu B, Fisher LW, Robey PG, Shi S. SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003; 100:5807-12. doi: 10.1073/pnas.0937635100.
Sakai VT, Zhang Z, Dong Z, Neiva KG, Machado MA, Shi S, Santos CF, Nor JE. SHED differentiate into functional odontoblasts and endothelium. J Dent Res. 2010; 89:791-6. doi: 10.1177/0022034510368647.
Chadipiralla K, Yochim JM, Bahuleyan B, Huang CY, Garcia‐Godoy F, Murray PE, Stelnicki EJ. Osteogenic differentiation of stem cells derived from human periodontal ligaments and pulp of human exfoliated deciduous teeth. Cell Tissue Res. 2010; 340:323-33. doi: 10.1007/s00441‐010‐0953‐0.
Osathanon T, Nowwarote N, Manokawinchoke J, Pavasant P. bFGF and JAGGED1 regulate alkaline phosphatase expression and mineralization in dental tissue‐derived mesenchymal stem cells. J Cell Biochem. 2013; 114:2551-61. doi: 10.1002/jcb.24602.
Nowwarote N, Pavasant P, Osathanon T. Role of endogenous basic fibroblast growth factor in stem cells isolated from human exfoliated deciduous teeth. Arch Oral Biol. 2015; 60:408-15. doi: 10.1016/j.archoralbio.2014.11.017.
Sukarawan W, Peetiakarawach K, Pavasant P, Osathanon T. Effect of Jagged‐1 and Dll‐1 on osteogenic differentiation by stem cells from human exfoliated deciduous teeth. Arch Oral Biol. 2016; 65:1-8. doi: 10.1016/j.archoralbio.2016.01.010.
Govitvattana N, Osathanon T, Taebunpakul S, Pavasant P. IL‐6 regulated stress‐induced Rex‐1 expression in stem cells from human exfoliated deciduous teeth. Oral Dis. 2013; 19:673-82. doi: 10.1111/odi.12052.
Nakamura S, Yamada Y, Katagiri W, Sugito T, Ito K, Ueda M. Stem cell proliferation pathways comparison between human exfoliated deciduous teeth and dental pulp stem cells by gene expression profile from promising dental pulp. J Endod. 2009; 35:1536-42. doi: 10.1016/j.joen.2009.07.024.
Yamaza T, Kentaro A, Chen C, Liu Y, Shi Y, Gronthos S, Wang S, Shi S. Immunomodulatory properties of stem cells from human exfoliated deciduous teeth. Stem Cell Res Ther. 2010; 1:5. doi: 10.1186/scrt5.
Dominici M, Le Blanc K, Mueller I, Slaper‐Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy. 2006; 8:315-7. doi: 10.1080/14653240600855905.
Kerkis I, Caplan AI. Stem cells in dental pulp of deciduous teeth. Tissue Eng Part B Rev. 2012; 18:129-38. doi: 10.1089/ten.TEB.2011.0327.
Jeon M, Song JS, Choi BJ, Choi HJ, Shin DM, Jung HS, Kim SO. In vitroand in vivocharacteristics of stem cells from human exfoliated deciduous teeth obtained by enzymatic disaggregation and outgrowth. Arch Oral Biol. 2014; 59:1013-23. doi: 10.1016/j.archoralbio.2014.06.002.
Bakopoulou A, Leyhausen G, Volk J, Tsiftsoglou A, Garefis P, Koidis P, Geurtsen W. Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth. Calcif Tissue Int. 2011; 88:130-41. doi: 10.1007/s00223‐010‐9438‐0.
Zheng Y, Liu Y, Zhang CM, Zhang HY, Li WH, Shi S, Le AD, Wang SL. Stem cells from deciduous tooth repair mandibular defect in swine. J Dent Res. 2009; 88:249-54. doi: 10.1177/0022034509333804.
Seo BM, Sonoyama W, Yamaza T, Coppe C, Kikuiri T, Akiyama K, Lee JS, Shi S. SHED repair critical‐size calvarial defects in mice. Oral Dis. 2008; 14:428-34.
Yamada Y, Nakamura S, Ito K, Sugito T, Yoshimi R, Nagasaka T, Ueda M. A feasibility of useful cell‐based therapy by bone regeneration with deciduous tooth stem cells, dental pulp stem cells, or bone‐marrow‐derived mesenchymal stem cells for clinical study using tissue engineering technology. Tissue Eng Part A. 2010; 16:1891-900. doi: 10.1089/ten.TEA.2009.0732.
Cordeiro MM, Dong Z, Kaneko T, Zhang Z, Miyazawa M, Shi S, Smith AJ, Nor JE. Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth. J Endod. 2008; 34:962-9. doi: 10.1016/j.joen.2008.04.009.
Rosa V, Zhang Z, Grande RH, Nor JE. Dental pulp tissue engineering in full‐length human root canals. J Dent Res. 2013; 92:970-5. doi: 10.1177/0022034513505772.
Kim S, Shin SJ, Song Y, Kim E. In vivoexperiments with dental pulp stem cells for pulp‐dentin complex regeneration. Mediators Inflamm. 2015; 2015:409347. doi: 10.1155/2015/409347.
Shi S, Bartold PM, Miura M, Seo BM, Robey PG, Gronthos S. The efficacy of mesenchymal stem cells to regenerate and repair dental structures. Orthod Craniofac Res. 2005; 8:191-9. doi: 10.1111/j.1601‐6343.2005.00331.x.
Huang GT, Yamaza T, Shea LD, Djouad F, Kuhn NZ, Tuan RS, Shi S. Stem/progenitor cell‐mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivomodel. Tissue Eng Part A. 2010; 16:605-15. doi: 10.1089/ten.TEA.2009.0518.
Gronthos S, Brahim J, Li W, Fisher LW, Cherman N, Boyde A, DenBesten P, Robey PG, Shi S. Stem cell properties of human dental pulp stem cells. J Dent Res. 2002; 81:531-5.
Wang J, Wang X, Sun Z, Wang X, Yang H, Shi S, Wang S. Stem cells from human‐exfoliated deciduous teeth can differentiate into dopaminergic neuron‐like cells. Stem Cells Dev. 2010; 19:1375-83. doi: 10.1089/scd.2009.0258.
Heng BC, Lim LW, Wu W, Zhang C. An overview of protocols for the neural induction of dental and oral stem cells in vitro. Tissue Eng Part B Rev. 2016; 22:220-50. doi: 10.1089/ten.TEB.2015.0488.
Jarmalaviciute A, Tunaitis V, Strainiene E, Aldonyte R, Ramanavicius A, Venalis A, Magnusson KE, Pivoriunas A. A new experimental model for neuronal and glial differentiation using stem cells derived from human exfoliated deciduous teeth. J Mol Neurosci. 2013; 51:307. doi: 10.1007/s12031‐013‐0046‐0
Fujii H, Matsubara K, Sakai K, Ito M, Ohno K, Ueda M, Yamamoto A. Dopaminergic differentiation of stem cells from human deciduous teeth and their therapeutic benefits for Parkinsonian rats. Brain Res. 2015; 1613:59-72. doi: 10.1016/j.brainres.2015.04.001.
Majumdar D, Kanafi M, Bhonde R, Gupta P, Datta I. Differential neuronal plasticity of dental pulp stem cells from exfoliated deciduous and permanent teeth towards dopaminergic neurons. J Cell Physiol. 2016; 231:2048-63. doi: 10.1002/jcp.25314.
Taghipour Z, Karbalaie K, Kiani A, Niapour A, Bahramian H, Nasr‐Esfahani MH, Baharvand H. Transplantation of undifferentiated and induced human exfoliated deciduous teeth‐derived stem cells promote functional recovery of rat spinal cord contusion injury model. Stem Cells Dev. 2012; 21:1794-802. doi: 10.1089/scd.2011.0408.
Bento LW, Zhang Z, Imai A, Nor F, Dong Z, Shi S, Araujo FB, Nor JE. Endothelial differentiation of SHED requires MEK1/ERK signaling. J Dent Res. 2013; 92:51-7. doi: 10.1177/0022034512466263.
Zhang Z, Nor F, Oh M, Cucco C, Shi S, Nor JE. Wnt/beta‐catenin signaling determines the vasculogenic fate of postnatal mesenchymal stem cells. Stem Cells. 2016; 34:1576-87. doi: 10.1002/stem.2334.
Zhang N, Chen B, Wang W, Chen C, Kang J, Deng SQ, Zhang B, Liu S, Han F. Isolation, characterization and multi‐lineage differentiation of stem cells from human exfoliated deciduous teeth. Mol Med Rep. 2016; 14:95-102. doi: 10.3892/mmr.2016.5214.
Kushnerev E, Shawcross SG, Hillarby MC, Yates JM. High‐plasticity mesenchymal stem cells isolated from adult‐retained primary teeth and autogenous adult tooth pulp—a potential source for regenerative therapies? Arch Oral Biol. 2016; 62:43-8. doi: 10.1016/j.archoralbio.2015.11.009.
Nowwarote N, Sukarawan W, Pavasant P, Osathanon T. Basic fibroblast growth factor regulates rex1 expression via il‐6 in stem cells isolated from human exfoliated deciduous teeth. J Cell Biochem. 2016. doi: 10.1002/jcb.25807.
Alipour R, Adib M, Masoumi Karimi M, Hashemi‐Beni B, Sereshki N. Comparing the immunoregulatory effects of stem cells from human exfoliated deciduous teeth and bone marrow‐derived mesenchymal stem cells. Iran J Allergy Asthma Immunol. 2013; 12:331-44.
Liu Y, Chen C, Liu S, Liu D, Xu X, Chen X, Shi S. Acetylsalicylic acid treatment improves differentiation and immunomodulation of SHED. J Dent Res. 2015; 94:209-18. doi: 10.1177/0022034514557672.
Silva Fde S, Ramos RN, de Almeida DC, Bassi EJ, Gonzales RP, Miyagi SP, Maranduba CP, Sant’Anna OA, Marques MM, Barbuto JA, Camara NO, da Costa Maranduba CM. Mesenchymal stem cells derived from human exfoliated deciduous teeth (SHEDs) induce immune modulatory profile in monocyte‐derived dendritic cells. PLoS One. 2014; 9:e98050. doi: 10.1371/journal.pone.0098050.
Ornitz DM, Itoh N. Fibroblast growth factors. Genome Biol. 2001; 2:Reviews 3005.
Dvorak P, Hampl A. Basic fibroblast growth factor and its receptors in human embryonic stem cells. Folia Histochem Cytobiol. 2005; 43:203-8.
Osathanon T, Nowwarote N, Pavasant P. Basic fibroblast growth factor inhibits mineralization but induces neuronal differentiation by human dental pulp stem cells through a FGFR and PLCgamma signaling pathway. J Cell Biochem. 2011; 112:1807-16. doi: 10.1002/jcb.23097.
Sukarawan W, Nowwarote N, Kerdpon P, Pavasant P, Osathanon T. Effect of basic fibroblast growth factor on pluripotent marker expression and colony forming unit capacity of stem cells isolated from human exfoliated deciduous teeth. Odontology. 2014; 102:160-6. doi: 10.1007/s10266‐013‐0124‐3.
Kim J, Park JC, Kim SH, Im GI, Kim BS, Lee JB, Choi EY, Song JS, Cho KS, Kim CS. Treatment of FGF‐2 on stem cells from inflamed dental pulp tissue from human deciduous teeth. Oral Dis. 2014; 20:191-204. doi: 10.1111/odi.12089.
Li B, Qu C, Chen C, Liu Y, Akiyama K, Yang R, Chen F, Zhao Y, Shi S. Basic fibroblast growth factor inhibits osteogenic differentiation of stem cells from human exfoliated deciduous teeth through ERK signaling. Oral Dis. 2012; 18:285-92. doi: 10.1111/j.1601‐0825.2011.01878.x.
Chaussain C, Eapen AS, Huet E, Floris C, Ravindran S, Hao J, Menashi S, George A. MMP2‐cleavage of DMP1 generates a bioactive peptide promoting differentiation of dental pulp stem/progenitor cell. Eur Cell Mater. 2009; 18:84-95.
Gorin C, Rochefort GY, Bascetin R, Ying H, Lesieur J, Sadoine J, Beckouche N, Berndt S, Novais A, Lesage M, Hosten B, Vercellino L, Merlet P, Le‐Denmat D, Marchiol C, Letourneur D, Nicoletti A, Vital SO, Poliard A, Salmon B, Muller L, Chaussain C, Germain S. Priming dental pulp stem cells with fibroblast growth factor‐2 increases angiogenesis of implanted tissue‐engineered constructs through hepatocyte growth factor and vascular endothelial growth factor secretion. Stem Cells Transl Med. 2016; 5:392-404. doi: 10.5966/sctm.2015‐0166.
Jarvinen E, Salazar‐Ciudad I, Birchmeier W, Taketo MM, Jernvall J, Thesleff I. Continuous tooth generation in mouse is induced by activated epithelial Wnt/beta‐catenin signaling. Proc Natl Acad Sci U S A. 2006; 103:18627-32. doi: 10.1073/pnas.0607289103.
Liu F, Chu EY, Watt B, Zhang Y, Gallant NM, Andl T, Yang SH, Lu MM, Piccolo S, Schmidt‐Ullrich R, Taketo MM, Morrisey EE, Atit R, Dlugosz AA, Millar SE. Wnt/beta‐catenin signaling directs multiple stages of tooth morphogenesis. Dev Biol. 2008; 313:210-24. doi: 10.1016/j.ydbio.2007.10.016.
Liu F, Millar SE. Wnt/beta‐catenin signaling in oral tissue development and disease. J Dent Res. 2010; 89:318-30. doi: 10.1177/0022034510363373.
Han N, Zheng Y, Li R, Li X, Zhou M, Niu Y, Zhang Q. beta‐catenin enhances odontoblastic differentiation of dental pulp cells through activation of Runx2. PLoS One. 2014; 9:e88890. doi: 10.1371/journal.pone.0088890.
Heo JS, Lee SY, Lee JC. Wnt/beta‐catenin signaling enhances osteoblastogenic differentiation from human periodontal ligament fibroblasts. Mol Cells. 2010; 30:449-54. doi: 10.1007/s10059‐010‐0139‐3.
Wang J, Liu B, Gu S, Liang J. Effects of Wnt/beta‐catenin signalling on proliferation and differentiation of apical papilla stem cells. Cell Prolif. 2012; 45:121-31. doi: 10.1111/j.1365‐2184.2012.00806.x.
Yang Y, Ge Y, Chen G, Yan Z, Yu M, Feng L, Jiang Z, Guo W, Tian W. Hertwig’s epithelial root sheath cells regulate osteogenic differentiation of dental follicle cells through the Wnt pathway. Bone. 2014; 63:158-65. doi: 10.1016/j.bone.2014.03.006.
Scheller EL, Chang J, Wang CY. Wnt/beta‐catenin inhibits dental pulp stem cell differentiation. J Dent Res. 2008; 87:126-30.
Nemoto E, Koshikawa Y, Kanaya S, Tsuchiya M, Tamura M, Somerman MJ, Shimauchi H. Wnt signaling inhibits cementoblast differentiation and promotes proliferation. Bone. 2009; 44:805-12. doi: 10.1016/j.bone.2008.12.029.
Rattanawarawipa P, Pavasant P, Osathanon T, Sukarawan W. Effect of lithium chloride on cell proliferation and osteogenic differentiation in stem cells from human exfoliated deciduous teeth. Tissue Cell. 2016; 48:425-31. doi: 10.1016/j.tice.2016.08.005.
Osathanon T, Pavasant P, Giachelli CM. Notch signaling biomaterials and tissue regeneration. In: Zhang LG, Khademhosseini A, Webster TJ, editors. Tissue and organ regeneration: advances in micro‐ and nanotechnology. Pan Stanford Publishing; Singapore. 2014. p. 535-63. doi: 10.1201/b15595‐18.
Zanotti S, Canalis E. Notch and the skeleton. Mol Cell Biol. 2010; 30:886-96. doi: 10.1128/MCB.01285‐09.
Tien AC, Rajan A, Bellen HJ. A Notch updated. J Cell Biol. 2009; 184:621-9. doi: 10.1083/jcb.200811141.
Mitsiadis TA, Graf D, Luder H, Gridley T, Bluteau G. BMPs and FGFs target Notch signalling via jagged 2 to regulate tooth morphogenesis and cytodifferentiation. Development. 2010; 137:3025-35. doi: 10.1242/dev.049528.
Lovschall H, Tummers M, Thesleff I, Fuchtbauer EM, Poulsen K. Activation of the Notch signaling pathway in response to pulp capping of rat molars. Eur J Oral Sci. 2005; 113:312-7. doi: 10.1111/j.1600‐0722.2005.00221.x.
Zhang C, Chang J, Sonoyama W, Shi S, Wang CY. Inhibition of human dental pulp stem cell differentiation by Notch signaling. J Dent Res. 2008; 87:250-5.
Wang X, He F, Tan Y, Tian W, Qiu S. Inhibition of Delta1 promotes differentiation of odontoblasts and inhibits proliferation of human dental pulp stem cell in vitro. Arch Oral Biol. 2011; 56:837-45. doi: 10.1016/j.archoralbio.2011.02.006.
He F, Yang Z, Tan Y, Yu N, Wang X, Yao N, Zhao J. Effects of Notch ligand Delta1 on the proliferation and differentiation of human dental pulp stem cells in vitro. Arch Oral Biol. 2009; 54:216-22. doi: 10.1016/j.archoralbio.2008.10.003.
Peetiakarawach P, Osathanon, T., Pavasant, P., Sukarawan, W. Effect of Jagged‐1 and Delta‐like‐1 on the proliferation of primary deciduous pulp cells. SWU Dent J. 2014; 7(Suppl):58-64.
Tonder KJ, Kvinnsland I. Micropuncture measurements of interstitial fluid pressure in normal and inflamed dental pulp in cats. J Endod. 1983; 9:105-9. doi: 10.1016/S0099‐2399(83)80106‐X.
Heyeraas KJ, Berggreen E. Interstitial fluid pressure in normal and inflamed pulp. Crit Rev Oral Biol Med. 1999; 10:328-36.
Paphangkorakit J, Osborn JW. The effect of normal occlusal forces on fluid movement through human dentine in vitro. Arch Oral Biol. 2000; 45:1033-41.
Hata M, Naruse K, Ozawa S, Kobayashi Y, Nakamura N, Kojima N, Omi M, Katanosaka Y, Nishikawa T, Naruse K, Tanaka Y, Matsubara T. Mechanical stretch increases the proliferation while inhibiting the osteogenic differentiation in dental pulp stem cells. Tissue Eng Part A. 2013; 19:625-33. doi: 10.1089/ten.tea.2012.0099.
Cai X, Zhang Y, Yang X, Grottkau BE, Lin Y. Uniaxial cyclic tensile stretch inhibits osteogenic and odontogenic differentiation of human dental pulp stem cells. J Tissue Eng Regen Med. 2011; 5:347-53. doi: 10.1002/term.319.
Yu V, Damek‐Poprawa M, Nicoll SB, Akintoye SO. Dynamic hydrostatic pressure promotes differentiation of human dental pulp stem cells. Biochem Biophys Res Commun. 2009; 386:661-5. doi: 10.1016/j.bbrc.2009.06.106.
Govitvattana N, Osathanon T, Toemthong T, Pavasant P. IL‐6 regulates stress‐induced REX‐1 expression via ATP‐P2Y1 signalling in stem cells isolated from human exfoliated deciduous teeth. Arch Oral Biol. 2015; 60:160-6. doi: 10.1016/j.archoralbio.2014.09.008.
Inoue T, Sugiyama M, Hattori H, Wakita H, Wakabayashi T, Ueda M. Stem cells from human exfoliated deciduous tooth‐derived conditioned medium enhance recovery of focal cerebral ischemia in rats. Tissue Eng Part A. 2013; 19:24-9. doi: 10.1089/ten.TEA.2011.0385.
Yamamoto A, Sakai K, Matsubara K, Kano F, Ueda M. Multifaceted neuro‐regenerative activities of human dental pulp stem cells for functional recovery after spinal cord injury. Neurosci Res. 2014; 78:16-20. doi: 10.1016/j.neures.2013.10.010.
Mita T, Furukawa‐Hibi Y, Takeuchi H, Hattori H, Yamada K, Hibi H, Ueda M, Yamamoto A. Conditioned medium from the stem cells of human dental pulp improves cognitive function in a mouse model of Alzheimer’s disease. Behav Brain Res. 2015; 293:189-97. doi: 10.1016/j.bbr.2015.07.043.
Sakai K, Yamamoto A, Matsubara K, Nakamura S, Naruse M, Yamagata M, Sakamoto K, Tauchi R, Wakao N, Imagama S, Hibi H, Kadomatsu K, Ishiguro N, Ueda M. Human dental pulp‐derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro‐regenerative mechanisms. J Clin Invest. 2012; 122:80-90. doi: 10.1172/JCI59251.